During the late 20th century a new biomedical platform, flow cytometry, appeared that revolutionized the field of hematopathology. It dramatically increased the speed of leukocyte subset analysis. The new platform was able to analyze cells or particles while they are in motion. The technology replaced visual counting with a microscope using epifluorescent illumination. Traditional data collection required about five minutes to identify 300 cells. Flow cytometers collects multi-parameter data at a rate of 5,000 cells per second. Sample data files contain at least 100,000 cell/particle events.
A flow cytometer as shown in FIG. 5, operates with an aqueous laminar focused flow column. The interrogation of particles occurs through a window perpendicular to the flow stream. Light scattered by cells/particles is captured as they flow in single file. While in flight, two kinds of optical signals are processed, 1) scattered light as it is related to morphology (i.e., size, volume, granularity) and 2) fluorescent signals that are specific to leukocyte subsets of interest. Normally, narrow wavelength lasers are used as illumination sources. To capture signals, sets of optical detectors with barrier filters and dichroic mirrors are arranged around the column. This permits digital fluorescence data collection over a specific narrow emission wavelength range. Flow cytometry data is managed by a dedicated software system that correlates the light scatter data with the various spectral channels that serve as leukocyte subset identifiers.
The current generation of clinical flow cytometers can correlate and display data from fifteen or more different light scatter and spectral channels. Having this many fixed channels requires a complex optical path of filters, mirrors and a PMT (photomultiplying tube) for each fluorescent channel. Moreover, separating this many spectral channels presents a significant challenge since the spectral properties of fluorescent dyes used to label cells tend to spill over into more than one channel, complicating subset separation.